The agr radiation: an early event in the evolution of staphylococci

Abstract

Agr is a global regulatory system in the staphylococci, operating by a classical two-component signaling module and controlling the expression of most of the genes encoding extracellular virulence factors. As it is autoinduced by a peptide, encoded within the locus, that is the ligand for the signal receptor, it is a sensor of population density or a quorum sensor and is the only known quorum-sensing system in the genus. agr is conserved throughout the staphylococci but has diverged along lines that appear to parallel speciation and subspeciation within the genus. This divergence has given rise to a novel type of interstrain and interspecies cross-inhibition that represents a fundamental aspect of the organism's biology and may be a predominant feature of the evolutionary forces that have driven it. We present evidence, using a newly developed, luciferase-based agr typing scheme, that the evolutionary divergence of the agr system was an early event in the evolution of the staphylococci and long preceded the development of the nucleotide polymorphisms presently used for genotyping. These polymorphisms developed, for the most part, within different agr groups; mobile genetic elements appear also to have diffused recently and, with a few notable exceptions, have come to reside largely indiscriminately within the several agr groups.

FIG. 1.

The staphylococcal agr operon. A) The agr locus is composed of two divergent transcriptional units driven by the P2 and P3 promoters. Sequence diversity in the variable region, comprised of the last one-third of agrB, agrD, and the first half of agrC, has generated the four specificity groups in S. aureus. AgrB is a transmembrane protein involved in the processing and secretion of AgrD to form the mature AIP, which is the ligand for AgrC. AgrC and AgrA form a two-component signaling team, which activates transcription from the P2 and P3 promoters, resulting in autoinduction and expression of RNAIII, an mRNA with global regulatory properties. Inhibition of the RNAIII transcription occurs via competitive binding of a noncognate AIP, which binds to the sensor domain of the AgrC receptor. B) Reporter strains with specificity for one of the four AIPs produced by S. aureus. The reporter strains are uncoupled for AIP production and sensation. Each strain is inactivated for agrBD and does not produce autoinducer. Exogenous AIP triggers the signaling cascade through the group-specific AgrCA two-component signaling module to activate the luxABCD reporter from the P3 promoter (55). Intergroup exchange of the receptor domain of AgrC (residues 1 to 205) has been shown to determine AIP specificity (56), and replacement of this region with other AgrC-class homologues could generate reporter sets with different autoinducer specificities.

FIG. 2.

Bioluminescent S. aureus agr typing assay. Strains representing each of the four standard agr groups, RN6734 (agr-I), RN6607 (agr-II), RN8465 (agr-III), and RN4850 (agr-IV), plus an agr-null control are streaked radially. The agr-specific reporter strains (RN9688, RN9689, RN9690, and RN9691), each carrying a group-specific AgrCA two-component signaling pair plus an agrp₃-lux fusion, are streaked alongside the standard strains, each of which activates one of the reporters, according to the specificity of its AIP. Reporters are identified by roman numerals designating their specificity group. Note that there is weak cross-reactivation of the group I reporter by the standard group IV strain and no activation by the agr-null. Bioluminescence was detected with a Hamamatsu CCD camera and is presented as a pseudocolor image, with the color bar indicating the signal intensity in counts. An AIP diffusion gradient is created on solid media (inset). As a representative example, the agr-II specific reporter strain (RN9689) was inoculated at graded distances from AIP-II producer RN6607 (agr-II). The bioluminescent signal generated from agrp₃-lux decreases as the distance between the reporter strain and the AIP producing strain increases.

FIG. 3.

The agr radiation. Evolutionary divergence of the agr locus has generated four agr specificity groups in S. aureus and one or more specificity groups among the non-aureus staphylococci. At left is a phylogenetic tree constructed from multiple sequence alignments of the AgrC variable receptor domain by CLUSTAL V. A tree constructed from 16S rRNA sequences has strikingly similar topology (data not shown) (7). The S. aureus agr lineages have given rise to the major MLST types observed today (http://www.mlst.net). There is evidence that putative mobile genetic elements (MGEs) are transferred horizontally among all agr types, but significant agr-specific predilections indicate that this lateral gene flow is constrained by some mechanism that may or may not be related to agr function. Lowercase letters in the diagram indicate examples of clinically significant MRSA isolates (labeled with their corresponding SCCmec type) and isolates whose genomes have been sequenced (*) (a, Berlin; b, EMRSA-15; c, Irish-1; d, 8325-4*; e, EMRSA-1; f, COL*; g, MSSA476*; h, MW2; i, MRSA252*; j, Mu50*; k, EMRSA-3; l, N315*).